Humans have a limited ability to regenerate tissue that is lost due to injury or disease. A greater understanding of the processes by which regeneration can occur could substantially improve medical approaches toward human organ and spinal cord injury, degenerative disease and loss of digits or limbs. This proposed research will investigate the function of a single developmental signaling pathway during regeneration in a simple animal model, the sea anemone, Nematostella vectensis. Specifically, Wnt/ss-catenin signaling will be investigated. Since key embryonic signaling pathways are active in regenerating tissue, it seems quite likely that Nematostella utilizes a similar suite of pathways during both embryogenesis and regeneration. Taken together, this leads to the hypothesis that the Wnt/ss-catenin pathway, which drives oral-aboral (O/A) specification during embryogenesis of Nematostella, also drives O/A identity during regeneration. Several predictions arise from this hypothesis: 1) During regeneration ss-catenin will be localized to the cytoplasm in aboral tissue and translocated to the nucleus in oral tissue 2) The axial identity of regenerating tissue will be controlled by the intracellular localization of ss-catenin; and 3) That signaling through ss-catenin activates transcriptional programs that drive fate specification of regenerating tissue. The overall goal of the proposed research is to determine the role of Wnt/ss-catenin signaling in axial patterning of regenerating tissue in the sea anemone Nematostella by completing the following specific aims: Specific Aim 1: To determine the intracellular localization of ss-catenin during regeneration of oral and aboral tissue using a ss-catenin polyclonal antibody. Specific Aim 2: To determine whether nuclear translocation of ss-catenin is necessary and/or sufficient to specify O/A identity in regenerating tissue by perturbing Wnt/ss-catenin signaling with RNAi and small molecule inhibitors of pathway components to inhibit and/or induce nuclear translocation of ss-catenin during regeneration. Specific Aim 3: To identify genes regulated by ss-catenin signaling in the production of oral and aboral tissue during regeneration. Following perturbation of Wnt/ss-catenin signaling, regulation of Wnt/ss-catenin responsive genes will be accessed via in situ hybridization and high throughput sequencing of the transcriptome in perturbed vs. non-perturbed (control) anemones. PUBLIC HEALTH RELEVANCE: The proposed project will expand our understanding of the molecular mechanisms underlying regeneration. Increased knowledge of how organs and tissue regenerate bears direct relevance to public health issues such as human organ and spinal cord injury, degenerative disease, and loss of digits or limbs.